The human retina is part of the central nervous system and, both developmentally and phenotypically, the retina shares the recalcitrance of brain and spinal cord with respect to functional repair. This is unfortunate in that, among heritable conditions alone, there are many examples of diseases involving the loss of retinal neurons. For example, retinitis pigmentosa, age-related macular degeneration and diabetic retinopathy are diseases characterized by the progressive death of light sensing photoreceptor cells of the retina. These diseases are the leading causes of incurable blindness in the western world, and are increasing rapidly in the developing Eastern world.
Since the intrinsic regenerative capacity of the human retina is extremely limited, a promising potential therapy for these diseases currently in research is a focus on cellular replacement. One strategy for replacing these cells has been to transplant retinal tissue from healthy donors to the retina of the diseased host. While the results of such studies have been encouraging in terms of graft survival, the problem of integration between graft and host has proved daunting. Laboratory studies have focused on multipotent stem cells (also variously referred to as progenitor cells, immature cells, undifferentiated cells or proliferative cells) for transplantation and differentiation. Proliferative stem or progenitor cells have been isolated from the hippocampus in laboratory animals, cultured and transplanted into various sites within the central nervous system (CNS) to subsequently differentiate into neurons and glial cells. Similarly, adult hippocampal cells have been shown to be capable of migrating into, and differentiating within, the mature dystrophic retina.
The isolation of true stem cells from the neuroretina, particularly cells able to differentiate into functional photoreceptor cells both in vitro and in vivo, has proven elusive. Putative retinal stem cells derived from the ciliary marginal zone pigment epithelial layer are described in U.S. Pat. No. 6,117,675. While these cells are said to be capable of proliferating in the absence of growth factor, there is no evidence that these cells are capable of integrating into a host retina and differentiating into functional mature cells in vivo.
Commonly assigned U.S. Pat. No. 7,514,259 is directed to neuroretina-derived photoreceptor cells which are capable of repopulating a human retina. These cells are derived from neural retinal tissue by removing the ciliary marginal zone and the optic nerve to eliminate contamination, and can be obtained from post-natal tissue.
Apart from difficulties involving the identification of viable human retinal progenitor cells, there are significant limitations involving the ability to culture these cells. Although such cells posses the capacity to survive, to differentiate into retinal neurons, and to integrate within the dystrophic host retina following transplantation, these cells have limited proliferative capacity. This represents a clear distinction between human retinal progenitor cells and other less restricted undifferentiated cell types, such as embryonic stem cells or induced pluripotent stem cells, which may not share such limitations.
For instance, and following isolation, human retinal progenitor cells can only be passaged a maximum number of seven (7) times in vitro without loss of multipotency, including the ability to proliferate in vivo and to form mature retinal cell types. This greatly limits the number of cells that can be obtained from a single isolate, the number of transplants that can be performed from a single cell isolation, and the further clinical application of these cells.
Attempts to immortalize fetal human retinal cells using SV40 transfection have not proven successful since the cells fail to express the markers of mature differentiated cells after transplantation. Similarly, other methods for culturing cells, such as the conditional immortalization or downregulation of pRb, as described for Muller glial cell lines expressing retinal stem cell genes, also yield cells which fail to differentiate into photoreceptors.
A variety of stem cell types have included, inter alia, the use of low oxygen culture conditions. See, for instance, U.S. Pat. No. 6,759,242 and U.S. Pat. No. 6,610,540 which relate to the enhanced differentiation of CNS precursor cells and neural crest stem cells under low oxygen culture conditions.
In view of the aforementioned, as well as the importance of human retinal progenitor cells for clinical evaluation and use, it will readily be appreciated that a need exists to improve the ability of such cells to reproduce in vitro while maintaining multipotency properties in vivo. These and other objectives of the invention will be clear from the following description.